We propose three specific aims that use fission yeast as a model system with which to deepen our understanding of the interactions between a mitotic chromosome and its spindle fiber. Strains of yeast that are mutant in kinetochore components, such as motor enzymes, microtubule-associated proteins, and regulatory kinases, will be studied in living cells with a sensitive 4-D light microscope to assess the role of particular kinetochore components in chromosome-spindle fiber attachment. Electron microscopy will be used in combination with tomography and immuno-localization to map known kinetochore components onto the slender, kinetochore-microtubule connections that have just been discovered. This work will use material preserved by rapid freezing to maximize the likelihood of getting reliable information about kinetochore structure. We will also use a set of laser tweezers developed over the last four years to carry out mechanochemical studies on the interactions between kinetochores and microtubules in vitro. Wild type and mutant chromosomes will be affixed to glass cover slips and then bound to microtubules that can attach a micro bead suitable for grasping with the laser. One set of experiments will characterize kinetochore-microtubule interactions in wild type fission yeast with respect to the strength and dynamics of their attachment. Another set will assess the impact of kinetochore dissection by genetics, measuring the effects of removing or altering specific components by mutation. A third set will assess the effects of changes in the medium surrounding the kinetochore on the physical properties of its connection with microtubules. Through these experiments we will evaluate the roles of specific kinetochore and microtubule-associated proteins on the connections that are responsible for accurate chromosome segregation at mitosis. The results from our work will inform biomedical science about important drug targets for cancer chemotherapy and genes that are significant for minimizing the risk of aneuploidy in both mitosis and meiosis. [unreadable] [unreadable]